USE OF DIFFERENTIAL GENE EXPRESSION
ANALYSIS TO STUDY SOMACLONAL VARIATION
IN OIL PALM
Fabienne Morcillo
1, Frédérique Richaud
1, Caroline Hartmann
2,
Alain Rival
1, Yves Duval
1& James W. Tregear
11GeneTrop Lab.,CIRAD-CP/IRD, PO. Box 5045, 34032 Montpellier Cedex 01, France.
2 University of Paris-XI, Plant Biotechnology Institute, CNRS ERS 569, Bat. 630, F-91405 Orsay Cedex, France.
Introduction
A
B
Micropropagation of oil palm by somatic embryogenesis provides a useful means of exploiting elite genotypes of this economically important species. Clonal progeny obtained by this approach have however been found to contain a variable percentage (averaging 5-10%) of palms bearing the mantled floral abnormality [1] (Fig. 1). The latter involves an overall feminisation of both male and female flowers: in the latter, the staminodes (vestigial stamens) develop as pseudocarpel structures which may develop after fertilisation to give mantled fruit. The most severely affected palms produce sterile flowers; thus the oil production of the plantation is reduced. Several key features of the mantled abnormality reveal that it is epigenetic in nature. Firstly, it has been observed that reversion to a normal floral phenotype may occur over a period of years following the onset of flowering. Secondly, although the mantled abnormality is strongly transmitted through tissue culture, only a weak non-Mendelian transmission of the mantled character is observed via seeds. Thirdly, the fact that the abnormal phenotype is unlikely to have resulted from a classical genetic mutation is borne out by ploidy, RAPD and AFLP studies previously carried out in our lab [5, 4]. Experimental Approaches
Given that the mantled character is epigenetic in nature, we have used an experimental approach based on differential display analysis [3] as a means of identifying genes which are differentially expressed in a mantled-related fashion (Fig. 2).
Results
Figure 3 summarises the data obtained thus far for
potential markers of the mantled abnormality identified by
differential display RT-PCR. Starting from a total of 46
differential bands observed on differential display gels, 13
markers have been validated by Northern hybridisation, of
which 6 correspond to genes which are more highly
expressed in normal tissues and 7 to genes which are more
highly expressed in abnormal tissues. Figure 4 shows a
Northern hybridisation performed to determine the tissue
specificity of one of the markers producing a stronger signal
in abnormal tissues. In this case it can be seen that
differential expression occurs not only in shoot apex
segments, but also in callus, in which the differential
expression pattern is even more marked.
Conclusions
The availability of cDNAs corresponding to genes which are differentially
regulated with respect to the mantled abnormality will allow us to study
the molecular mechanisms by which tissue culture stress eventually
leads to the reprogramming of gene expression in the oil palm flower.
Recent studies have shown that the mantled abormality is associated
with genomic DNA hypomethylation [2] and it will be interesting to study
this phenomenon for specific genes and their promoter sequences. From
a practical point of view, it is hoped that the availability of mantled
markers will allow the refinement of tissue culture regeneration protocols
and/or the establishment of a clonal conformity test. This in turn will
allow the use of somatic embryogenesis as a large scale
micropropagation method for oil palm.
References
[1] Corley RHV, Lee CH, Law LH, Wong CY: The Planter, Kuala Lumpur 62: 233-240 (1986). [2] Jaligot E, Rival A, Beule T, Dussert S, Verdeil J-L: Plant Cell Rep. 7: 684-690 (2000). [3] Liang P, Pardee AB: Science 257: 967-971 (1992).
[4] Rival A, Bertrand L, Beulé T, Combes MC, Trouslot P, Lashermes P: Plant Breeding 117: 73-76 (1998). [5] Rival A, Beulé T, Barre P, Hamon S, Duval Y, Noirot M: Plant Cell Rep. 16: 884-887 (1997).
Fig. 1. Morphology of normal and mantled oil palm flowers (A) and
fruits (B).
Fig. 4:Northern hybridisation analysis of the tissue specificity of a putative marker of abnormal tissues. Abbreviations: SPDC, normal seed palm-derived culture; NCDC, ACDC, abnormal (mantled) clone-derived culture; INFLO, normal or mantled inflorescence (N or A).
No of potential markers identified by differential display 46
No of individual cloned cDNA fragments obtained 58
No of cDNAs producing differential signals in Northern retesting
13
No of cDNAs producing stronger Northern signal in normal tissue
6
No of cDNAs producing stronger Northern signal in abnormal
tissue 7
Fig. 3.Summary of data obtained to date on differential display markers.
Normal culture Abnormal culture
DIFFERENTIAL DISPLAY ANALYSIS
Cloning and sequencing of potential marker cDNAs
Validation of markers using original normal and abnormal material
Characterisation of tissue specificity Testing on other genotypic backgrounds Isolation of full length cDNAs Protein studies PCR cloning of 5' flanking regions
Fig. 2.Experimental strategy.
Normal Mantled Normal ISPMB.VIth International Congress on Plant Molecular Biology. Québec June 2000 Mantled
